The purpose of this project is to better understand the regulation of iron homeostasis. Dysregulation of iron homeostasis results in iron overload or iron deficiency. The major regulator of iron homeostasis is the 25 amino acid peptide hepcidin. Hemochromatosis caused by genetic mutations in HFE, TfR2 and HJV is due primarily to dysregulation of hepcidin transcription. We have recently discovered that a serum protease encoded by the Tmprss6 gene is a potent downregulator of hepcidin. A robust transcriptional response to iron is only observed in intact animals. To understand normal transcriptional regulation of hepcidin by iron and also to gain insight into the roles of the products of the genes known to be involved in its regulation, we will investigate hepcidin transcription in liver, the primary site of synthesis in wildtype mice and mutant mice lacking these genes. In a two-pronged approach we will: 1) study nuclear extracts prepared from mice stimulated with iron;and 2) investigate the response of hepcidin promoter reporter constructs in an in vivo system that we have devised. Classical ex vivo techniques including EMSA, ChIP assays, DNA affinity purification and mass spectrophotometric identification will be employed to identify transcription factors in nuclear extracts. These studies will reveal which transcription factors are involved in the response to iron and how this response is modified in mutant animals. There is marked variability in the expression of the hemochromatosis phenotype in patients homozygous for the C282Y HFE mutation. Studies to date have failed to identify either genetic or environmental factors that account for any but a very small portion of the known variability. We will investigate, in mice, whether there is transgenerational epigenetic modification of iron absorption, based on the experience of prior generations with iron. Mice receiving iron-deficient diets or diets containing iron excess will be bred and their offspring will either be weaned to the same diet or to a normal diet. Those weaned to a deficient or iron excess diet will be bred for additional generations. If transgenerational epigenetic modification occurs, then the iron burden of iron deficient mice moved to normal diet will be greater than that of mice derived from iron-heavy progenitors. PUBLIC HEALTH RELEVANCE: By studying various mouse models of iron overload and iron deficiency, we will gain an understanding how the body regulates iron. This will open the door to treatments of iron overload, the anemia of chronic inflammation, as well as of hemochromatosis itself. If we are able to demonstrate that the regulation of iron absorption is inherited epigenetically, this finding would not only explain the variable penetrance of hemochromatosis, but would greatly alter our perception of how natural selection functions over the span of only a few generations.